Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi

Kickstein, Eva; Harms, Klaus; Wackernagel, Wilfried

doi:10.1099/mic.0.2007/005256-0

Cited by 38 publications

(64 citation statements)

References 76 publications

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“…1b). In previous experiments the recBCD mutation decreased transformation more strongly with chromosomal trp + DNA (fivefold; Kickstein et al, 2007). We confirmed the stronger effect with genomic DNA fragments carrying a bla (Ap-resistance) gene inserted in the benK gene giving Ap-resistant transformants fivefold less frequently in recBCD In a recD mutant (strain EK6) the HFIR transformation frequency was identical to that in the wild-type (Fig.…”

Section: Resultssupporting

confidence: 67%

“…The bacterial strains and plasmids used in this study are listed in Table 1. The A. baylyi strains JV28 ('wild-type') and its derivatives KOM18 (DrecBCD) and EK6 (DrecD) have been described previously (de Vries et al, 2003;Kickstein et al, 2007). The strain KOM17 (DsbcCD) was constructed by allelic exchange as described for the generation of the recBCD deletion strain .…”

Section: Methodsmentioning

confidence: 99%

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The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

Harms

Wackernagel

2008

Microbiology

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During natural transformation of Acinetobacter baylyi, the genomic integration of foreign (nonhomologous) DNA is possible when the DNA contains a single segment homologous to the recipient genome (anchor) through homologous recombination in the anchor facilitating illegitimate recombination in the neighbouring foreign DNA (homology-facilitated illegitimate recombination; HFIR). DNA integration by HFIR occurs about 10 000 times less frequently than fully homologous recombination, but at least 100 000-fold more frequently than integration in the absence of any homology. We investigated the influence of the RecBCD enzyme (DNase/ helicase) and SbcCD DNase (DNA-structure-specific single-strand endonuclease and exonuclease) on HFIR. In a recBCD null mutant the acquisition of foreign DNA was elevated 11-fold relative to wild-type cells by a 6.9-fold increased HFIR frequency and by the integration of longer stretches of foreign DNA in each event. In an sbcCD null mutant, the foreign DNA acquisition was 4.5-fold higher than in the wild-type, while homologous transformation with large DNA molecules was unaffected and increased 3.2-fold with small DNA fragments. The sbcCD mutation partially suppressed the high UV sensitivity and low viability of the recBCD mutant and also decreased its foreign DNA acquisition by HFIR to the lower level of the sbcCD mutant. We propose that suppression of HFIR results from the elimination of double-stranded intermediates of the HFIR process during transformation by RecBCD, and by SbcCD interfering with branched molecules. Our results provide evidence that the homologous recombination enzymes RecBCD and SbcCD control the level of foreign DNA acquisition by HFIR.

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Section: Resultssupporting

confidence: 67%

Section: Methodsmentioning

confidence: 99%

The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

Harms

Wackernagel

2008

Microbiology

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“…For a complementation study, the pgaR gene was PCR amplified from chromosomal DNA of P. gallaeciensis DSM 17395 using the primers 5Ј-ATGGCAACCAAAGTTAATCTTGATC-3Ј and 5Ј-TTAAATGAT GATCAGCCCGAGAC-3Ј and cloned into the EcoICRI sites of pRK415iq (27), resulting in pRKpgaR. The open reading frame of pgaR and the lac promoter were PCR amplified from pRKpgaR using the primers 5Ј-CCAATACGCAAA CCGCCTCTC-3Ј and 5Ј-TTAAATGATGATCAGCCCGAGAC-3Ј and ligated into the EcoICRI site of pBBR1MCS-2 (28), giving rise to plasmid pBB0808.…”

Section: Fig 1 (A and B)mentioning

confidence: 99%

Tropodithietic Acid Production in Phaeobacter gallaeciensis Is Regulated by N-Acyl Homoserine Lactone-Mediated Quorum Sensing

Berger

Neumann

Schulz

et al. 2011

Journal of Bacteriology

110

145

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The production of N-acyl homoserine lactones (AHLs) is widely distributed within the marine Roseobacter clade, and it was proposed that AHL-mediated quorum sensing (QS) is one of the most common cell-to-cell communication mechanisms in roseobacters. The traits regulated by AHL-mediated QS are yet not known for members of the Roseobacter clade, but production of the antibiotic tropodithietic acid (TDA) was supposed to be controlled by AHL-mediated QS in Phaeobacter spp. We describe here for the first time the functional role of luxR and luxI homologous genes of an organism of the Roseobacter clade, i.e., pgaR and pgaI in Phaeobacter gallaeciensis. Our results demonstrate that the AHL synthase gene pgaI is responsible for production of N-3-hydroxydecanoylhomoserine lactone (3OHC 10 -HSL). Insertion mutants of pgaI and pgaR are both deficient in TDA biosynthesis and the formation of a yellow-brown pigment when grown in liquid marine broth medium. This indicates that in P. gallaeciensis the production of both secondary metabolites is controlled by AHLmediated QS. Quantitative real-time PCR showed that the transcription level of tdaA, which encodes an essential transcriptional regulator for TDA biosynthesis, decreased 28-and 51-fold in pgaI and pgaR genetic backgrounds, respectively. These results suggest that both the response regulator PgaR and the 3OHC 10 -HSL produced by PgaI induce expression of tdaA, which in turn positively regulates expression of the tda genes. Moreover, we confirmed that TDA can also act as autoinducer in P. gallaeciensis, as previously described for Silicibacter sp. strain TM1040, but only in the presence of the response regulator PgaR.

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“…DNA end resection deficiency has a significant impact on bacterial cell viability and growth: E. coli recBC recJ (39,40) and B. subtilis addAB ⌬recJ double mutants (10) are deeply affected, and the Acinetobacter baylyi ⌬recBCD ⌬recJ double mutant is not viable (41).…”

Section: Resultsmentioning

confidence: 99%

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

et al. 2014

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bHomologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. Cells are under constant genotoxic pressure from both endogenous and exogenous sources. DNA damage needs to be repaired to avoid the formation of deleterious mutations, abortion of replication, and lethal chromosomal breakage. Homologous recombination (HR) is a crucial mechanism that repairs a variety of DNA lesions, including DNA double-strand breaks (DSBs), single-strand DNA gaps, and interstrand cross-links.DSBs are probably the most deleterious DNA damage that a cell can encounter. They are induced in cells by physical agents such as ionizing radiation or UV light, chemical agents, and natural products such as mitomycin C (MMC) or bleomycin. DSBs also result from replication fork collapse during chromosomal replication (1). The failure to repair DSBs can lead to cell death and, in the case of disrepair, can trigger large-scale chromosome rearrangements, favoring the generation of genetic diversity.In bacteria, DSBs are for the most part processed through HR, which requires a homologous DNA template to carry out faithful repair of the damaged DNA duplex. In intensively replicating cells (vegetative growth phase) or immediately after the passing of the replication fork, the sister chromatid can be used as an intact template. In nonreplicating phases, such as late stationary phase or in spores (which contain a single copy of the chromosome), DSBs are more likely repaired by an illegitimate repair pathway. Indeed, illegitimate recombination (IR) does not require an intact homologous sequence but as a consequence has reduced fidelity.The involvement of DSB repair by HR in a range of fundamental cellular processes (e.g., chromosome integrity, replication, segregation, etc.) reveals that HR is conserved in all living organisms. The initiating step of the repair mechanism consists of the resection of the DSB...

show abstract

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi

Cited by 38 publications

References 76 publications

The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

Tropodithietic Acid Production in Phaeobacter gallaeciensis Is Regulated by N-Acyl Homoserine Lactone-Mediated Quorum Sensing

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

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